Exposure to Freeze–Thaw Conditions Increases Virulence of

Article Cite This: Environ. Sci. Technol. 2018, 52, 14180−14186

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Exposure to Freeze−Thaw Conditions Increases Virulence of Pseudomonas aeruginosa to Drosophila melanogaster Arsham Hakimzadeh,†,‡ Mira Okshevsky,† Vimal Maisuria,† Eric Deź iel,‡ and Nathalie Tufenkji*,† †

Department of Chemical Engineering, McGill University, 3610 University Street, Montréal, Québec H3A 0C5, Canada INRS-Institut Armand-Frappier, 531 boulevard des Prairies, Laval, Québec H7V 1B7, Canada

Environ. Sci. Technol. 2018.52:14180-14186. Downloaded from pubs.acs.org by UNIVERSITE DE SHERBROOKE on 01/11/19. For personal use only.

‡

ABSTRACT: Groundwater contamination by pathogenic bacteria present in land-applied manure poses a threat to public health. In cold climate regions, surface soil layers experience repeated temperature fluctuations around the freezing point known as freeze−thaw (FT) cycles. With global climate change, annual soil FT cycles have increased, and this trend is expected to continue. It is therefore of interest to understand how FT cycles impact soil microbial communities. This study investigates the influence of FT cycles on the growth, culturability, biofilm formation, and virulence of the bacterial opportunistic pathogen Pseudomonas aeruginosa, a ubiquitous bacterium found in soil and water, responsible for infections in immunocompromised hosts. Our findings demonstrate that exposure to FT had no significant effect on growth or culturability of the bacteria. However, FT treatment significantly increased biofilm formation and delayed the onset of swimming motility, factors that are important for the pathogenicity of P. aeruginosa. An in vivo study using a chronic infection model revealed an increase in the virulence of P. aeruginosa after FT exposure. These results suggest that the impact of climate change on natural FT cycles may be affecting the ecology of soil-borne pathogens and host−pathogen interactions in unexpected ways.

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INTRODUCTION Microorganisms in frozen surface soils can survive for several months, and can be carried from their original location by spring melt.1,2 Controlled laboratory and field studies show that microorganisms can migrate significant distances in both vertical and horizontal directions,3 subsequently decreasing the quality of groundwater.3−8 The microbial community of lakes, which are also used as drinking water sources, is seeded from the surrounding catchment area of the lake.9−11 There is therefore a direct link between pathogens present in soil and the possible contamination of drinking water systems.8 In cold climate regions such as Canada, the northeastern United States, and Scandinavian countries, microorganisms in the surface layers of soil experience temperature fluctuations around the freezing point of water, referred to as freeze−thaw (FT) cycles.12−16 This kind of temperature stress influences ecosystem carbon and nutrient cycling and disrupts the natural aggregate formation of soil.17 During winter, ice formed on the surface of the soil works as an insulation layer and protects the soil and microorganisms living in it from extreme temperature fluctuations.18 With global climate change, this insulation layer has become thinner in winter, and over the past 50 years, the frequency of annual soil FT cycles has increased, with this trend projected to continue.19 Bacteria subjected to repeated FT cycles behave differently than bacteria maintained at a constant cold temperature. For example, exposure to repeated FT cycles can increase cell surface hydrophobicity and © 2018 American Chemical Society

compromise the viability and swimming motility of Yersinia enterocolitica by reducing the expression of flagellin-encoding genes f lhD and f liA.20 FT exposure can significantly suppress biofilm formation of Bacillus subtilis, even while bacterial growth rates remain unchanged.21 Exposure to increased numbers of FT cycles can increase the mobility of Salmonella typhimurium in water-saturated sand-packed columns.22 Controlled laboratory studies show that exposure to FT can significantly impact the survival, motility, and biofilm formation of bacterial pathogens present in soil.20−25 These are factors that may have an impact on the virulence of bacterial cells.24,25 Yet, the impact of FT exposure on the virulence of soil-borne opportunistic pathogens has never been examined. Understanding how exposure to FT affects pathogenic bacteria present in soil is important for the appropriate assessment of pathogen activity in soil and aquatic environments, and also for the development of effective drinking water protection strategies. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is ubiquitous in soil, freshwater, and other moist environments.26−28 Unlike some other waterborne pathogens, P. aeruginosa is highly resilient and able to adapt to a wide Received: Revised: Accepted: Published: 14180

August 31, 2018 November 7, 2018 November 16, 2018 November 16, 2018 DOI: 10.1021/acs.est.8b04900 Environ. Sci. Technol. 2018, 52, 14180−14186

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Figure 1. Temperature profile used for FT treatment and control condition (10 °C).

range of temperatures and nutrient-limited environments.29 It can cause acute and chronic infections in immunocompromised hosts, burn and surgical patients, and individuals suffering from cystic fibrosis.30−33 This pathogen can contaminate drinking water34 and has been responsible for numerous outbreaks of infection from environmental sources.28 The chronic infectivity of P. aeruginosa is enhanced by its ability to form biofilms,35 which are surface-attached communities of bacterial cells displaying increased resistance to the host immune response and recalcitrance to antibiotic treatment.30 The fruit fly Drosophila melanogaster is commonly used as an alternative animal model to study host−pathogen interactions.36,37 The fruit fly is a simple to handle and inexpensive animal model that has a well-studied innate immune system consisting of both cellular and humoral responses.36,38 The fly feeding assay used here models a chronic microbial infection,37 in which the subjects are infected and monitored for mortality. This study investigates the impact of FT cycles on the growth, culturability, biofilm formation, motility, and virulence of the model organism P. aeruginosa PA14. This microorganism was chosen because it is an extensively well-studied pathogenic strain, relevant to the behavior of environmental P. aeruginosa strains due to the high degree of genomic homogeneity between strains of this species.39 Bacteria were subjected to multiple FT cycles for 5 consecutive days,20,21 during which temperature fluctuations mimicked the natural temperature profile experienced in upper layers of soil during spring melt in southern Canada (0 ± 10 °C).20,21,40,41 Key bacterial behaviors from a public health perspective were investigated, and the effects of a nutrient-rich medium versus no nutrients were compared. Experiments were not conducted in soil specifically due to the complexity of such a medium. To reduce the possibility of confounding effects and to be certain only one variable was being changed at a time, experiments were conducted in controlled liquid media. The two extremes of nutrient availability were tested so that any effects linked to this variable could be easily identified. The virulence of P. aeruginosa was investigated using an in vivo assay of chronic infection on D. melanogaster. We conclude that FT exposure can substantially increase biofilm formation and virulence of P. aeruginosa, while bacterial growth, culturability, and survival remain unaffected.

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glycerol solution prior to experiments. Frozen cultures were streaked onto LB agar plates and grown overnight at 37 °C. For each experiment, a single colony was inoculated into 25 mL of LB broth in a 125-mL Erlenmeyer flask and incubated at 37 °C and 180 rpm. After overnight growth, cells were washed by centrifugation at 5000g for 10 min and resuspended in 25 mL of LB or 100 mM KCl solution. This washing procedure was repeated twice. Finally, the bacterial suspension was diluted to OD600 = 0.3 (≈ 6.0 × 108 CFU/mL), and was immediately used for experiments. Freeze−Thaw (FT) Treatment. Five mL of diluted bacterial suspension described above was dispensed into a sterile glass tube with a loosely fitted plastic cap to ensure aerobic conditions. Glass tubes were immediately placed at 10 °C (control condition) or in a thermocycler (Julabo CORIO CD-200FCORIO CD-200F/Heating Circulator) for FT treatment. The FT thermocycler was programmed to simulate a temperature range that could reasonably be expected to occur during spring in Québec, Canada. The temperature ramp rate during FT exposure was chosen based on environmentally relevant conditions in southern Québ ec, as reported previously.20,21 The temperature ranged between −10 °C and +10 °C and consisted of four stages, controlled by the Julabo GmbH EasyTemp version 3.8.1 software. Starting from +10 °C, the temperature decreased to −10 °C at a constant rate of 2.5 °C/h for 8 h, until −10 °C was reached. Then, the temperature stayed at −10 °C for 4 h before gradually increasing from −10 °C to +10 °C over 8 h, again at a constant rate of 2.5 °C/h. Figure 1 illustrates this temperature profile. In the final stage, the temperature was held at +10 °C for 4 h. Total time of one FT cycle was 24 h. The bacteria remained in the FT thermocycler for 5 consecutive days to experience five FT cycles. Bacteria Culturability and Cell Viability. Different approaches were used to evaluate cell culturability/viability after 5 consecutive days of temperature treatment. First, the optical density of bacterial suspensions was measured at 600 nm, as one means to verify whether the bacteria were growing or lysing during this period (Tecan Infinite M200 Pro microplate reader, Switzerland). Second, bacterial culturability was evaluated by CFU counting. Bacterial suspensions were serially diluted in sterile PBS, and 100 μL of diluted suspensions was spread on LB agar plates and incubated at 37 °C for 18 h. Third, a membrane integrity assay was performed using the BacLight Cell Viability Kit (Invitrogen), with fluorescent stains SYTO 9 and propidium iodide (PI) prepared as per manufacturer’s instructions. Briefly, following 5 days of temperature treatment, 1 mL of bacterial suspension was centrifuged in a microcentrifuge tube (≈ 4.0 × 107 CFU),

MATERIALS AND METHODS

Bacterial Strain and Growth. Lysogeny broth (LB) and potassium chloride (KCl) were purchased from Fisher Scientific (Canada). Pseudomonas aeruginosa PA14 (UCBPPPA14) was kept as stock culture at −80 °C in a 30% (v/v) 14181

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materials.21,42,43 In this study, culturability and bacterial cell wall integrity of P. aeruginosa were determined using CFU counting and BacLight Live/Dead assay, respectively. Figure 2

the supernatant was discarded, and the pellet was resuspended in 50 μL of SYTO 9 and 50 μL of PI with the final concentrations of 6 μM and 30 μM, respectively, and incubated for 15 min in the dark. Fluorescent bacteria were imaged using a Zeiss 800 confocal laser scanning microscope. ImageJ 1.47v software was used to quantify the relative percentage of biomass comprised of intact cells (green) and cells with compromised cell membranes (red). Finally, bacterial growth curves were obtained for cells exposed to the different temperature treatments. Bacteria were diluted to OD600 = 0.03 in LB, then 200 μL of this bacterial suspension was dispensed in 96-well microtiter plates (Falcon, Corning, Fisher Scientific Canada) and incubated at 37 °C for 24 h in a Tecan Infinite M200 Pro microplate reader. OD600 was measured every 30 min. The initial optical density of the suspension was subtracted from each time point. Biofilm Formation and Swimming Motility Assay. Biofilm formation of bacteria was quantified using the crystal violet (CV) assay. After temperature treatment, bacteria were diluted in LB to OD600 = 0.03 and 100 μL of diluted suspension was dispensed into flat-bottom, 96-well microtiter plates (Falcon, Corning, Fisher Scientific Canada). Plates were incubated at 37 °C for 3, 6, 9, 12, 18, and 24 h under static conditions. At these time points, the suspension was discarded and wells were rinsed three times with 150 μL of sterile water to remove planktonic bacteria. Biofilms were stained with 150 μL of 0.1% (w/v) CV, incubated for 10 min at room temperature, and rinsed at least three times with 200 μL of sterile water to remove the unattached CV solution. When the plate was completely dry, the dye was solubilized in 200 μL of 30% (v/v) acetic acid (Glacial Certified ACS, Fisher Chemical, Fisher Scientific Canada) and quantified at 550 nm using a Tecan Infinite M200 Pro microplate reader. Sterile LB and bacteria kept at a constant 10 °C were used as blank and control, respectively. Bacterial motility was studied after exposure of the cells to 5 consecutive days of FT or constant temperature. In both cases, 5 μL of cell suspension was stabbed in the middle of LB Petri dishes containing 0.2% (w/v) agar. Plates were incubated at 37 °C for 12 h and the diameter of the swimming halo was measured every 3 h. Infection of Drosophila melanogaster. Fruit flies (D. melanogaster) were infected orally in a fly feeding assay to study the chronic infection of P. aeruginosa. Briefly, 6- to 7-day old adult flies were anesthetized under a gentle stream of carbon dioxide and male flies were separated. Vials containing 5 mL of 5% sterile sucrose agar were prepared and 2.3-cm sterile filter paper disks were placed on the solidified agar. A volume of 100 mL (≈ 4.0 × 109 CFU) of bacteria exposed to FT cycles or constant 10 °C was centrifuged at 7000g for 3 min and the pellet was resuspended in 100 μL of sterile 5% sucrose and dispensed on the filter papers. Male adult flies were starved of food and water for 6 h prior to the experiments and 10 flies were transferred to each vial. Flies were kept in the incubator at 25 °C and 65% humidity. The lights inside the incubator were switched on and off to simulate 16 h of day and 8 h of night. Post-infection mortality of flies was monitored daily for 8 days, with each treatment tested twice, each time in triplicate.

Figure 2. Relative culturability (C/C0), membrane integrity, and optical density at 600 nm of P. aeruginosa, after 5 days of temperature treatment. Legend: FT LB = cells exposed to FT while suspended in LB; FT KCl = cells exposed to FT while suspended in 100 mM KCl; 10 °C LB = cells maintained at 10 °C while suspended in LB; and 10 °C KCl = cells maintained at 10 °C while suspended in 100 mM KCl. Results show mean value ± standard deviation for three replicates. Statistical analysis using Student’s t test with p = 0.05 shows no significant difference between temperature treatments.

shows the results of OD600 measurements, CFU counts relative to initial CFUs (C/C0), and membrane integrity assays following 5 days of FT, or 5 days at 10 °C (the cells were suspended in LB or 100 mM KCl during the temperature treatment). The OD600 of bacterial suspensions was not altered by 5 days of FT treatment. During these 5 days, the culturability of P. aeruginosa decreased from an initial value of 6.0 × 108 CFU/mL (before temperature treatment) by a factor of ∼10 following all temperature treatments (FT LB, FT KCl, 10 °C LB, and 10 °C KCl). However, membrane integrity results show that following all temperature treatments, more than 80% of the cells had intact cell membranes. The discrepancy between these results could be explained by a portion of cells which have not lost membrane integrity, but are not able to replicate and form coloniesthe so-called “viable but non-culturable” (VBNC) state.20,21,44,45 Exposure to environmental stresses such as high or low temperature, starvation, oxidation, or chemical stress can induce the VBNC state.46 For example, the VBNC state in P. aeruginosa can be induced by copper ions at concentrations relevant to drinking water supply systems.47,48 Traditional detection methods fail to differentiate between VBNC and dead cells.49 Nonetheless, these cells can maintain their virulence in some cases50 and pose a potential threat to public health.49 For instance, a study using Caenorhabditis elegans shows that the virulence of VBNC and culturable Listeria monocytogenes does not differ significantly.51 Li et al.46 reported that the morphology, physical and chemical resistances, metabolism, adhesion properties, and virulence of VBNC cells may differ from their culturable counterparts.46 Therefore, being in a VBNC state does not mean the cells have lost their ability to become virulent. It has been suggested that temperature decreases in aquatic environments are one of the most common inducers of the VBNC state in P. aeruginosa.52,53 We therefore propose that the decrease in temperature experienced by P. aeruginosa in our study induced the VBNC state in a subpopulation of cells in all treatments. Neither the growth rate nor the maximum OD600

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RESULTS AND DISCUSSION Effect of Temperature Treatment on Bacterial Survival. Freezing can negatively impact the permeability of bacterial cell walls due to the formation of ice crystals that disrupt cellular membranes and cause leakage of cellular 14182

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more pronounced over time up to 18 h after biofilm growth, most likely due to increasing cell numbers magnifying the FT effect. Temperature treatment in KCl resulted in less biofilm than treatment in full-nutrient media, which is consistent with our earlier observation that there is a slight lag in the onset of exponential growth of bacteria FT treated in KCl (Figure 3b). If under these conditions, fewer cells were immediately ready to replicate, the onset of biofilm formation would also be delayed. Because biofilm formation and planktonic swimming are distinct physiological states, we expected to observe a decrease in swimming motility corresponding to the increase in biofilm formation. Swimming assays revealed that FT treatment had no effect on the swimming speed (= distance/time) of P. aeruginosa (i.e., the slope of the line in Figure 5).

were significantly different between the four conditions after 24 h of incubation (Figure 3). However, there was a slight lag in

Figure 3. Growth curves of P. aeruginosa over 24 h. The optical density at the initial time point (t = 0) is subtracted from measurements at each time point. Legend: LB = cells exposed to FT or maintained at 10 °C while suspended in LB; KCl = cells exposed to FT or maintained at 10 °C while suspended in 100 mM KCl. Results represent mean value ± standard deviation. The graphs show mean ± standard deviation for six replicates. Statistical analysis using Student’s t test with p = 0.05 shows no significant difference between thermal treatments.

the onset of exponential growth of bacteria FT treated in KCl (Figure 3b), possibly indicating that under these conditions, fewer cells were immediately ready to replicate. KCl-treated cells have the additional challenge of maintaining cellular functions in a nutrient-deprived environment, in which the resources required to repair damaged cell membranes and maintain cell functions are scarce. We therefore conclude that although survival rates are unaffected by FT cycles, FT treated bacteria may be slower to reactivate under favorable conditions, with this effect exacerbated by a lack of nutrients. FT Exposure Leads to Increased Biofilm Formation. Biofilms are structured assemblages of bacterial cells that are recalcitrant to antibiotic treatment and the host immune system.54,55 Biofilm formation is an important mechanism by which P. aeruginosa is able to create and maintain chronic infections.54,56,57 We therefore decided to investigate the effect that temperature fluctuations have on biofilm formation of P. aeruginosa. Using the crystal violet assay, biofilm formation was measured at 3, 6, 9, 12, 18, and 24 h after temperature treatment had concluded. Although growth rate was not impacted by temperature treatment (Figure 3), we observed that FT treatment resulted in more biofilm formation than that of the control held at 10 °C (Figure 4). This difference became

Figure 5. Swimming motility of P. aeruginosa on LB plates with 0.2% agar. Migration diameter was measured every 3 h. The graphs show mean ± standard deviation for six replicates. Statistical analysis was done using Student’s t test and stars show the significant difference between FT and control condition at each time point (p < 0.05).

However, the onset of swimming was delayed in the case of FT treatment, as shown by a lag in the initial onset of swimming halo formation. This lag was observed for FT treatment in both LB and KCl, although cells that experience FT in LB media were able to recover over time (i.e., no significant difference in halo size was observed after 12 h in the case of LB treated bacteria). This initial delay in the onset of swimming is consistent with our previous observation that FT treatment can delay the onset of exponential growth (Figure 3). Other studies have shown that environmental stresses such as oxidative stress caused by chemicals,58 extreme temperatures,44 or exposure to ultraviolet light (UV),55 can cause P. aeruginosa to switch to the more stress-tolerant biofilm phenotype. Because the majority of chronic bacterial infections are associated with biofilms,59 an increase in biofilm formation following temperature fluctuations suggests that P. aeruginosa may be more adept at creating chronic infections following FT treatment. We tested this hypothesis using an in vivo chronic infection model. Exposure to FT Renders P. aeruginosa More Virulent. Fruit flies were fed temperature-treated P. aeruginosa for a period of 200 h, and the mortality of the flies was monitored for 8 days following the onset of exposure. The results indicate that FT-treated P. aeruginosa has higher virulence against D. melanogaster than the control bacteria held at 10 °C (Figure 6). Mortality rates of flies were higher if the infecting bacteria were temperature-treated in LB rather than KCl, which is consistent with our results suggesting FT treatment in KCl is more damaging to P. aeruginosa than FT treatment in LB. No significant mortality of flies was observed when the flies were exposed to 5% sterile sucrose agar without any bacteria, indicating that fly mortality can be directly attributed to P.

Figure 4. Effect of thermal treatment on biofilm formation of P. aeruginosa. The optical density at the initial time point (t = 0) is subtracted from measurements at each time point. Results represent mean value ± standard deviation. The graphs show mean ± standard deviation for six replicates. Significant differences between FT and control conditions at each time point are indicated by stars (p < 0.05). 14183

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nutrient content of soil has a role to play in modulating how FT cycles impact the virulence of soil-borne microorganisms. This is the first study to report the effect of FT on the virulence of a bacterial pathogen in an animal model; further studies are needed to explore the generality of this observation for other bacterial pathogens of importance to environmental and public health.

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AUTHOR INFORMATION

Corresponding Author

Figure 6. Virulence of P. aeruginosa toward D. melanogaster in a chronic infection model. Post-infection mortality was monitored for 8 days. Tests were done in six replicates, with 10 flies in each vial. Statistical analysis was done using Student’s t test and stars show the significant difference between FT and control condition (p < 0.05).

*Phone: (514) 398-2999; fax: (514) 398-6678; e-mail: [email protected]. ORCID

Nathalie Tufenkji: 0000-0002-1546-3441 Notes

aeruginosa infection. A control experiment was conducted to ensure that the number of viable bacteria capable of causing infections did not differ between treatments. CFU numbers were measured after centrifugation and resuspension in sucrose. No significant change in the number of cells was observed between different treatments (data not shown). Although CFU numbers at the start of fly exposure were the same, it is possible that a VBNC population of cells could “reactivate” during infection and contribute to the increased virulence of the FT-treated bacteria. In addition, increased virulence following FT treatment may be at least partially explained by the higher levels of biofilm formation35 by FTtreated P. aeruginosa cells. In many cases, the biofilm phenotype can be considered a stress response mechanism in which bacteria switch to a state more resistant to environmental stresses.60 An alternative explanation for the observed difference in fly mortality could be that the flies find the FTtreated bacteria more palatable, and therefore expose themselves to these bacteria more readily through their changes in behavior. However, a previous study has shown that when given a choice between virulent and harmless mutant strains of the same bacteria, D. melanogaster does not exhibit a preference.61 Therefore, we do not believe that FT exposure has made the bacteria more or less palatable to the flies. We suggest that the stress experienced by P. aeruginosa during multiple FT cycles triggers the biofilm phenotype, which contributes to increased virulence in a chronic infection model. FT affected P. aeruginosa will be expected to form more biofilm on soil particles; however, the bacteria that do end up in drinking water will likely be more virulent, nonetheless. Although no model is perfect, including the mouse, the fruit fly has been widely validated as a useful model to predict the potential of virulence in mammalian hosts.62,63 In particular, this is well shown for infection with P. aeruginosa.64,65 The stress experienced by P. aeruginosa following FT cycles may be exacerbated by a lack of nutrients (KCl treatment), however, this lack of nutrients did not result in higher virulence (Figure 6), most likely because FT-damaged cells were unable to repair themselves without adequate nutrients. Environmental Implications. The increased virulence observed in P. aeruginosa toward D. melanogaster after FT exposure suggests that increasing FT cycles brought on by global climate change have the potential to increase the virulence of opportunistic pathogens found in soil. This has important implications for public health and the safety of drinking water in cold areas. Although no natural soil will be as nutrient poor as a KCl solution, our results suggest that the

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Canada Research Chairs program, the Fonds de recherche du Québec − Nature et technologies (FRQNT), the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation, and the McGill Engineering Doctoral Award Program (MEDA). We also thank MarieChristine Groleau for her technical assistance with infection of D. melanogaster and helpful discussions.

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